The main objectives of the proposed studies are to isolate, chemically characterize, and assess the urinary bladder carcinogenicity of mutagenically active chemical(s) present in several solvent fractions of the carcinogenic plant bracken fern (BF); and to isolate and identify BF-derived mutagenic metabolites excreted in rat urine and cows milk. We have isolated three different classes of chemicals from BF. Some have shown mutagenic activity for Salmonella typhimurium TA 98 and/or TA 100, and displayed rodent local, bladder, and/or intestinal carcinogenicity. Isolated chemicals include tannin, the flavonols quercetin and kaempferol (present in BF as non-mutagenic glycones), and a third as yet uncharacterized chemical(s) that displays strong direct mutagenicity for TA 100, but not TA 98. This chemical is excreted into rat urine following BF feeding, and may be responsible for the principal bladder carcinogenic activity of BF. Our recent preliminary data suggest that this unknown chemical(s) is not a flavonol, and that it is not structurally related to any known bladder carcinogens. We hypothesize that this chemical(s) exists in BF as promutagen(s) that requires in vivo bioactivation, and that proximate biotransformation products are excreted in urine, feces, or milk of animals fed BF. Our studies will focus on a systematic evaluation, characterization, and biological assessment of those components in BF exhibiting these characteristics. Isolation procedures will be based on solvent extractions, and thin-layer, high pressure liquid, ion-exchange, and gas-liquid chromatographic procedures. Structural analyses will be by ultraviolet, proton magnetic resonance, mass and infrared spectrophotometric methods. Isolation procedure enhancement ratios will be monitored by mutagenic assays using TA 98 and TA 100 with and without "S-9" incubation. Short-term in vivo toxicity, presence of urinary mutagens, histopathologic acute effects on bladder, and induced bladder ornithine decarboxylase will also be used to monitor isolates. Carcinogenicity of isolated, active compounds will be determined by oral or subcutaneous administration to rats, or by direct intravesical implantation in mice. BF is a food delicacy consumed by humans in the USA and elsewhere. In Japan its use was associated with increased esophageal cancer risk. We isolated quercetin from BF and demonstrated its major (about 80%) intestinal and minor (about 20%) bladder oncogenicity. Other unidentified mutagens still are present in BF and may be responsible for its bladder carcinogenesis. These studies seek to identify these biologically active chemicals.